Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

Use at an assay dependent dilution. To detect mIL-1b by Western Blot analysis this antibody can be used at a concentration of 0.1 - 0.2 µg/ml. Used in conjunction with compatible secondary reagents the detection limit for recombinant mIL-1b is 1.5 - 3.0 ng/lane, under either reducing or non-reducing conditions.

ELISA

Use at an assay dependent dilution. To detect mIL-1b by direct ELISA (using 100µl/well antibody solution) a concentration of at least 0.5µg/ml of this antibody is required. This antigen affinity purified antibody, in conjunction with compatible secondary reagents, allows the detection of 0.2 - 0.4 ng/well of recombinant mIL-1b.

Neutralising

Use at an assay dependent dilution. To yield one-half maximal inhibition [ND50] of the biological activity of mIL-1b (50 pg/ml), a concentration of 100 - 150 ng/ml of this antibody is required.

Activation of the IL1B precursor involves a CASP1-catalyzed proteolytic cleavage. Processing and secretion are temporarily associated.

Cellular localization

Cytoplasm, cytosol. Lysosome. Secreted, exosome. Cytoplasmic vesicle, autophagosome. Secreted. The precursor is cytosolic. In response to inflammasome-activating signals, such as ATP for NLRP3 inflammasome or bacterial flagellin for NLRC4 inflammasome, cleaved and secreted. IL1B lacks any known signal sequence and the pathway(s) of its secretion is(are) not yet fully understood (PubMed:24201029). On the basis of experimental results, several unconventional secretion mechanisms have been proposed. 1. Secretion via secretory lysosomes: a fraction of CASP1 and IL1B precursor may be incorporated, by a yet undefined mechanism, into secretory lysosomes that undergo Ca(2+)-dependent exocytosis with release of mature IL1B (PubMed:15192144). 2. Secretory autophagy: IL1B-containing autophagosomes may fuse with endosomes or multivesicular bodies (MVBs) and then merge with the plasma membrane releasing soluble IL1B or IL1B-containing exosomes (PubMed:24201029). However, autophagy impacts IL1B production at several levels and its role in secretion is still controversial. 3. Secretion via exosomes: ATP-activation of P2RX7 leads to the formation of MVBs containing exosomes with entrapped IL1B, CASP1 and other inflammasome components. These MVBs undergo exocytosis with the release of exosomes. The release of soluble IL1B occurs after the lysis of exosome membranes (By similarity). 4. Secretion by microvesicle shedding: activation of the ATP receptor P2RX7 may induce an immediate shedding of membrane-derived microvesicles containing IL1B and possibly inflammasome components. The cytokine is then released in the extracellular compartment after microvesicle lysis (PubMed:11728343). 5. Release by translocation through permeabilized plasma membrane. This may occur in cells undergoing pyroptosis due to sustained activation of the inflammasome (By similarity). These mechanisms may not be not mutually exclusive.

Images

ab9722 staining IL1 beta in murine bone marrow-derived macrophages by Immunocytochemistry/ Immunofluorescence. Cells are immortalised murine bone marrow-derived macrophages stably transfected with GFP-LC3 (green) to visualise autophagosomes. The cells were fixed in paraformaldehyde, permeabilised in 0.01% Triton X-100 and then blocked using 5% serum for 1 hour at 20°C. Samples were then incubated with primary antibody at 1/200 for 1 hour at 20°C. The secondary antibody used was a goat anti-rabbit IgG conjugated to Alexa Fluor® 568 (red) used undiluted.

Tissue was fixed with paraformaldehyde and blocked with 10% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/100 in diluent) for 10 hours at 25°C. An AlexaFluor®555-conjugated donkey anti-goat IgG polyclonal (1/300) was used as the secondary antibody.

Cells were fixed with formaldehyde, permeabilized with 0.1% Triton X-100 + 3% BSA and blocked with 3% BSA for 3 hours at 25°C. Samples were incubated with primary antibody (1/100 in 1% BSA in PBS) for 1 hour at 25°C. A FITC-conjugated goat anti-rabbit polyclonal IgG (1/100) was used as the secondary antibody. Nuclei were stained with DAPI (blue).

IHC image of IL1 beta staining in mouse kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9722, 1µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

The Il-1 beta antibody ab9722 is tested for reactivity against recombinant mature active IL-1 beta. We do not have any in-house data demonstrating reactivity with the precursur/pro-form, but we at least predict reactivity with the denatured pro-form, g...